The conjugation of biomolecules to solid and gel supports is a common operation in may laboratories, and many methods have been developed for this purpose. Immobilization of enzymes (I. Chernukhin, E. Klenova, Anal. Biochem. 2000, 280:178–81), oligonucleotides (J. Andreadis; L. Chrisey, Nucleic Acids Res. 2000, 28:e5; A. Drobyshev et al., Nucl. Acids. Res. 1999, 27:4100–4105), antibodies (P. Soltys, M. Etzel, Biomaterials 2000, 21:37–48), and antigens (M. Oshima, M. Atassi, Immunol. Invest. 1989, 18:841–851) on solid and gel supports enables the preparation of useful products such as chromatographic media (Meth. Enzym. W. Jakoby, M. Wilchek, eds., 1974, 34, Academic Press, NY), catalysts (T. Krogh et al., Anal. Biochem., 1999, 274:153–62), biosensors (J. Spiker, K. Kang, Biotechnol. Bioeng. 1999, 66:158–63), and numerous diagnostic (G. Ramsay, Nature Biotechnol., 1998, 16:40–44) and research tools (C. Bieri et al., Nature Biotechnol. 1999, 17:1105–1108). Even whole cells may be immobilized by such methods (E. Olivares, W. Malaisse, Int. J. Mol. Med. 2000, 5:289–290).
The most robust form of attachment of a biomolecule to a surface or other support is via covalent bonds. Typically, such bonds are heteroatom-based (e.g., amide, ester, and disulfide bonds), because such bonds are easily formed under mild conditions. Non-covalent attachment via specific binding pairs (e.g., biotin-avidin or antibody-antigen interactions) is also commonly employed, but such methods still require initial conjugation of the specific binding pairs to the biomolecule and support. The use of carbon-carbon bonds for this purpose is very rare, because formation of C—C bonds is more difficult, especially under the mild aqueous conditions appropriate for working with proteins.
The use of the Diels-Alder reaction to attach a member of a specific binding pair has been described. In this report (M. N. Yousaf and M. Mrksich, J. Am. Chem. Soc., 1999, 121:4286), a Diels-Alder reaction was used to covalently attach a biotinylated diene to an immobilized dienophile, and the immobilized biotin was subsequently used to non-covalently immobilize streptavidin. Thee workers have more recently used a Diels-Alder reaction to immobilize the peptide RGDS on a self-assembled alkanethiol monolayer on a gold surface (M. N. Yousaf, B. T. Houseman, M. Mrksich, Angew. Chem. Int. Ed. Engl., 2001, 40:1093). The use of the Dials-Alder reaction to effect the actual covalent coupling or immobilization event of large biomolecules, however, had not previously been described.
The conjugation of biomolecules to one another is likewise a very common procedure, and is subject to most of the concerns and limitations described above for biomolecule immobilization. Covalent attachment of haptens to proteins has been a target of synthetic endavors since the discovery by Landsteiner that this process can convert non-immunogenic molecules to immunogenic material (K. Landsteiner, H. Lampl, Biochem. Zeitschr. 1918, 86:343). The application of this concept to carbohydrates by Goebel and Avery revealed that covalent carbohydrate-protein conjugates are immunogenic and can generate anti-carbohydrate antibodies (W. Goebel, J. Exp. Med. 1940, 72:33). The use of Landsteiner's principle has led to the development of carbohydrate-protein conjugates that are valuable tools in glycomedical research, and that are useful as pharmaceuticals. In particular, protein conjugates of fragments of the capsular polysaccharide of Haemophilus influenzae type b have become established as successful vaccines (J. Robbins et al., J. Am. Med. Assoc. 1996, 276:1181). Several other bacterial saccharide-protein conjugates are in various stages of clinical studies (E. Kondau et al., J. Infect. Dis. 1998, 177:383–387; E. Konadu et al., Infect. Immun. 2000, 68:1529–1534) while numerous others are in the pre-clinical phase (V. Pozagay et al., Proc. Natl. Acad. Sci. USA 1999, 96:5194).
The choice of methods for covalent bond formation between biomolecules such as carbohydrates and proteins is restricted by their limited solubility in organic solvents, and in many cases by their pH and temperature sensitivity. In almost all cases, water is the only solvent that can be used for conjugation of carbohydrates or proteins, and the conditions are usually limited to temperatures under 50° C. and pH values between 6 and 8.
Numerous methods have been developed for the attachment of polysaccharides to proteins (C. Peeters et al., in Vaccine Protocols, A. Robinson et al., Eds., 1996 Humana Press, NJ, p. 111; W. Dick, Jr., M. Beurret, in Contrib. Microbiol. Immunol., J. Cruse and R. Lewis, eds., 1989, 10:48–114, Karger, Basel; H. Jennings, R. Sood, in Neoglycoconjugates. Preparation and Applications. Y. Lee, R. Lee, eds., Academic Press, New York, 1994, p. 325). However, only a few of these methods are capable of coupling oligosaccharides to carriers in a site-selective fashion. Most prominent among these is reductive amination, which converts the reducing-end residue of the polysaccharide into a polyhydroxy alkylamino moiety, which unfortunately causes the loss of this unit as a true carbohydrate in the resulting glycoconjugate (V. Pozgay, Glycoconjugate J. 1993, 10:133).
This problem can be solved by chemical synthesis of oligosaccharide glycosides with aglycons that bear a (latent) reactive group. Examples include alkenyl groups (M. Nashed, Carbohydr. Res. 1983, 123:241–246; J. Allen, S. Danishefsky, J. Am. Chem. Soc. 1999, 121:10875), 3-aminopropyl (G. Veeneman et al., Tetrahedron 1989, 45:7433), 4-aminophenylethyl (R. Eby, Carbohydr. Res. 1979, 70:75), 4-aminophenyl (S. Stirm et al., Justus Liebigs Ann. Chem. 1966, 696:180), 6-aminohexyl (J. Hermans et al., Rec. Trav. Chim. Pays-Bas 1987, 106:498; R. Lee et al., Biochemistry 1989, 28:1856), 5-methoxycarbonylpentyl (S. Sabesan, J. Paulson, J. Am. Chem. Soc. 1986, 108:2068; V. Pozsgay, Org. Chem. 1998, 63:5983), 8-methoxycarbonyloctyl (R. Lemieux et al., J. Am. Chem. Soc. 1975, 97, 4076; B. Pinto et al., Carboydr. Res. 1991, 210, 199) 4-aminobenzyl (W. Goebel, J. Exp. Med. 1940, 72:33), ω-aldehydoalkyl (V. Pozsgay, Glycoconjugate J. 1993, 10:133), 3-(2-aminoethylthio)propyl (Y. Lee, R. Lee, Carbohydr. Res. 1974, 37:193), 2-chloroethylthiogly (M. Ticha et al., Glycoconjugate J. 1996, 13:681) and 1-O-succinimide derivatives (M. Andersson, S. Oscarson, Bioconjugate Chem. 1993, 4:246; B. Davis, J. Chem. Soc. Perkin I 1999, 3215).
These aglycons introduce spacers that can be linked to a protein either directly or after insertion of a secondary linker. For this purpose the use of an activated dicarboxylic acid has been reported (R. van den Berg et al., Eur. J. Org. Chem. 1999, 2593–2600). In another procedure, a sulfhydryl group at the terminal position of the spacer allows the formation of a disulfide bridge with proteins using the dithiopyridyl method (J. Evenberg et al., J. Infect. Dis. 1992, 165(sup. 1):S152). In a related protocol, a thiolated protein is coupled with a maleimido-derivatized saccharide (J. Mahoney, R. Schnaar, Methods Enzymol. 1994, 242:17). N-acryloylamidophenyl glycosides may be coupled to unmodified proteins using a Michael addition (A. Romanowska et al., Methods Enzymol. 1994, 242:90). As an alternative to glycoside formation, direct coupling of a carbohydrate to a linker via amide bonds has also been used (A. Fattom et al., Infect. Immun. 1992, 60:584–589), but this approach is limited to carboxyl-containing carbohydrates.
The yields of any of these methods rarely exceed 40% and are generally in the 10–20% range (R. van den Berg et al., Eur. J. Org. Chem. 1999, 2593–2600), especially when medium or high carbohydrate loading in the conjugate is attempted. This problem is compounded by the fact that the oligosaccharide haptens, usually obtained in multistep syntheses or by controlled degradation of polysaccharides, can rarely be reconverted in their active or activable form after the coupling procedure. An additional problem with most chemical coupling methods employed to date is the formation of cross-linked byproducts, due to the presence of multiple reactive functional groups (e.g., amines, acids, hydroxyls, and sulfhydryls) on most biomolecules. Avoidance of this problem requires that the reactive groups be blocked, with requires additional processing steps and may alter the physicochemical and immunological properties of the biomolecule. Thus, there remains a need for a mild and site-selective method for coupling biomolecules to one another, which avoids the problems of low yields, crosslinking, and loss of starting materials. For similar reasons there remains a need for mild and selective methods for attaching biomolecules to surfaces and solid and gel supports.